Intestinal epithelial dysfunction is one of the key factors in the pathogenesis of heat stress-induced disease. The purpose of this experiment was to investigate whether betaine protects IEC-6 cells from dysfunction induced by heat stress (HS) through antioxidative mechanism. The IEC-6 cells were divided into four groups: control group incubated at 37 °C, while those in heat treated groups (41 °C for 24 h) were pretreated with 0, 0.5 and 1 mmol/L betaine, respectively. Cell viability, apoptosis, barrier function protein and oxidative status were analyzed. Compared to control group, the rate of apoptosis and the Bax and caspase-3 expressions significantly increased in HS group (P < 0.05), however, cell activity, total antioxidative capacity (T-AOC), activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and the expression of Bcl-2, claudin-1 and occludin decreased significantly (P < 0.05). Betaine (0.5 mmol/L) can significantly enhance IEC-6 cell viability, while significantly reduce the apoptosis rate of cell during HS (P < 0.05). Meanwhile, the expression of Bcl-2, claudin-1 and occludin proteins were also significantly upregulated (P < 0.05) when compared to HS group. HS had a negative impact on IEC-6 cells, while betaine protected from damage caused by HS via increasing the antioxidative capacity. This suggested that betaine might be an effective dietary additive to protect animals from detrimental intestinal reactions caused by HS.